Optical sensor conjugates for detecting reactive oxygen and/or reactive nitrogen species in vivo

ABSTRACT

The present invention provides methods and compositions based on optical sensor conjugates that are useful for detecting reactive oxygen, reactive nitrogen, or both species that are a direct result of inflammation caused by tissue damage.

CROSS REFERENCE TO RELATED APPLICATION

This application is a 371 of International Application No.PCT/US2011/038903, filed Jun. 2, 2011, which claims priority from U.S.Provisional Application Ser No. 61/350,772 filed on Jun. 2, 2010, all ofwhich are incorporated herein by reference in their entireties.

STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

The present invention was made with government support Under Grant No.1-U01-HL080731 awarded by the National Institutes of Health. The UnitedStates Government has certain rights in the invention.

FIELD OF THE INVENTION

The present disclosure describes the design and synthesis of sensorconjugates for the detection of reactive oxygen and/or reactive nitrogenspecies.

BACKGROUND OF THE INVENTION

Inflammation is a ubiquitous response to acute or chronic tissue injury.Reactive oxygen and nitrogen species (ROS/RNS) generated duringinflammation are causal to or can exacerbate pathogenesis of Alzheimerdisease, atherosclerosis, cancer, ischemia-reperfusion injury in stroke,inflammatory bowel disease, myocardial infarction, and organtransplantation. Generation of the majority of ROS species is driven byheavy metal catalyzed oxidation reactions and enzymatically bymyeloperoxidase (MPO). MPO is a heme-containing enzyme that mediatesproduction of hypochlorious acid (HOCl/OCl⁻) from chloride ion (Cl⁻) andhydrogen peroxide (H₂O₂). MPO can accommodate and oxidize a number ofsmall molecule substrates that bind to the active site displacing watermolecules.

MPO has been shown to be a biomarker of myocardial infarction (MI) andcoronary artery disease, due to the central role of MPO-dependentprotein modification in the pathogenesis cardiovascular diseases. Inatherosclerosis, low density lipoprotein (LDL) particles are oxidized byHOCl, chloramines, phenoxyl radical intermediates, peroxynitrite (ONOO⁻)and MPO-dependent nitrogen dioxide (NO₂) production, driving lipid-ladenmacrophages to become atherosclerotic foam cells; the core of vulnerableplaques.

Imaging of MPO function and ROS generation in cells and animal models ofhuman diseases has been hampered by the lack of probes with appropriatepharmacokinetics, suitable emission wavelengths to overcome tissueauto-fluorescence, and adequate specificity for relevant ROS. Tailoringof optical imaging probes for certain ROS/RNS species have beenattempted with varied success for measuring nitric oxide,superoxide/.OH, H₂O₂, ONOO⁻, and HOCl. However, in vivo use of theseprobes is not practical, because most are small molecules withunfavorable washout kinetics, have poor dye properties, and/or areactivated by numerous ROS. Therefore, there exists a need for new waysfor monitoring ROS/RNS generation.

SUMMARY OF THE INVENTION

The present invention provides methods and compositions based on opticalsensor conjugates that can be used to detect ROS/RNS, such as HOCl/OCl—and ONOO—, that are the direct result of inflammation. The opticalsensor conjugates include a fluorogenic small molecule with a linkerconnecting the fluorogenic small molecule to a particle, e.g.,nanoparticle or microparticle. The fluorogenic small molecule can beoxidized in vivo by ROS/RNS causing the small molecule to be releasedfrom the linker and fluoresce. Thus, the fluorescent small molecule canbe detected, e.g., by microscopy.

Accordingly, in a first aspect the present invention provides, interalia, methods of detecting reactive oxygen, reactive nitrogen, or bothin a subject. The methods include administering to the subject anoptical sensor conjugate comprising a fluorogenic small molecule, aparticle or a polymer, and a linker that connects the small molecule tothe particle, for a time sufficient for the small molecule to react withthe reactive oxygen or reactive nitrogen, or both, thereby releasing thesmall molecule from the optical sensor conjugate and becomingfluorescent; and providing a fluorescence image of the subject with thesmall molecule, thereby imaging the subject.

In one embodiment, the small molecule with the linker is a compound ofFormula I:

wherein:

Z is the linker and comprises C₃₋₂₀ alkyl, wherein any of the carbons inC₃₋₂₀ alkyl can be replaced with —C(O)—, C(O)O—, —C(O)NR^(A)—, —C(NH)—,oxygen, sulfur, —SO₂—, —NR^(A)SO₂— —NR^(A)—;

X is selected from

N₃, C(O)Cl, and C(O)OR^(A);

Y is selected from O, S, Se, Te, N(R^(A)), and C(Me)₂;

R¹ is selected from H, C₁₋₆ alkyl, OR^(A), and NR^(C)R^(D), wherein saidC₁₋₆ alkyl is optionally substituted by OR^(A) or NR^(C)R^(D);

or 2 or 3 R¹ adjacent to each other and together with the C atoms towhich they are attached form 1 or 2 heterocycloalkyl, optionallysubstituted by 1, 2, 3, or 4 substituents independently selected from H,OH, C₁₋₆ alkyl, OR^(A), C(O)R^(B), C(O)NR^(C)R^(D), and C(O)OR^(A);

R^(A), R^(B), R^(C) and R^(D) are independently selected from H, C₁₋₆alkyl, and succinimidyl; and

m is 1, 2, 3, or 4.

In some embodiments, X is C(O)OR^(A) and Y is O.

In some embodiments, R¹ is NR^(C)R^(D).

In one embodiment, the invention provides a small molecule and linker,which is

In some embodiments, the particle further comprises a reporting agent.

In some embodiments, the fluorescence image is provided by fluorescencemicroscopy.

In some embodiments, the subject is a mammal.

In some embodiments, the reactive oxygen, reactive nitrogen, or both arecaused by and/or associated with inflammation.

In some embodiments, the inflammation is caused by and/or associatedwith tissue injury.

In some embodiments, the reactive oxygen, reactive nitrogen, or bothspecies are caused by and/or associated with Alzheimer's disease,atherosclerosis, cancer, stroke, inflammatory bowel disease, or organtransplantation.

In another aspect, the invention provides a composition for detectingreactive oxygen and reactive nitrogen species in a subject, thecomposition comprising: a fluorogenic small molecule, a particle, and alinker that connects the small molecule to the particle.

In some embodiments, the particle further comprises a reporting agent.

In some embodiments, the small molecule with the linker is a compound ofFormula I:

wherein:

Z is the linker and comprises C₃₋₂₀ alkyl, wherein any of the carbons inC₃₋₂₀ alkyl can be replaced with —C(O)—, C(O)O—, —C(O)NR^(A)—, —C(NH)—,oxygen, sulfur, —SO₂—, —NR^(A)SO₂— —NR^(A)—;

X is selected from

N₃, C(O)Cl, and C(O)OR^(A);

Y is selected from O, S, Se, Te, N(R^(A)), and C(Me)₂;

R¹ is selected from H, C₁₋₆ alkyl, OR^(A), and NR^(C)R^(D), wherein saidC₁₋₆ alkyl is optionally substituted by OR^(A) or NR^(C)R^(D);

or 2 or 3 R¹ adjacent to each other and together with the C atoms towhich they are attached form 1 or 2 heterocycloalkyl, optionallysubstituted by 1, 2, 3, or 4 substituents independently selected from H,OH, C₁₋₆ alkyl, OR^(A), C(O)R^(B), C(O)NR^(C)R^(D), and C(O)OR^(A);

R^(A), R^(B), R^(C) and R^(D) are independently selected from H, C₁₋₆alkyl, and succinimidyl; and

m is 1, 2, 3, or 4.

Definitions

The term “alkyl” refers to a hydrocarbon chain that may be a straightchain or branched chain, containing the indicated number of carbonatoms. For example, C₁-C₁₂ alkyl indicates that the group may have from1 to 12 (inclusive) carbon atoms.

The term “cycloalkyl” as employed herein includes saturated cyclic,bicyclic, tricyclic, or polycyclic hydrocarbon groups having 3 to 12carbons. Any ring atom can be substituted. The cycloalkyl groups cancontain fused rings. Fused rings are rings that share a common carbonatom. Examples of cycloalkyl moieties include, but are not limited to,cyclopropyl, cyclohexyl, methylcyclohexyl, adamantyl, and norbornyl.

The term “heterocyclyl” refers to a nonaromatic 3-10 memberedmonocyclic, 8-12 membered bicyclic, or 11-14 membered tricyclic ringsystem having 1-3 heteroatoms if monocyclic, 1-6 heteroatoms ifbicyclic, or 1-9 heteroatoms if tricyclic, said heteroatoms selectedfrom O, N, or S (e.g., carbon atoms and 1-3, 1-6, or 1-9 heteroatoms ofN, O, or S if monocyclic, bicyclic, or tricyclic, respectively). Theheteroatom may optionally be the point of attachment of the heterocyclylsubstituent. Any ring atom can be substituted. The heterocyclyl groupscan contain fused rings. Fused rings are rings that share a commoncarbon atom. Examples of heterocyclyl include, but are not limited to,piperidinyl, morpholino, pyrrolinyl, and pyrrolidinyl. The term,“compound,” as used herein is meant to include all stereoisomers,geometric iosomers, tautomers, and isotopes of the structures depicted.

The term “reactive oxygen species” or “ROS” refers to reactive moleculesthat contain the oxygen atom. ROS are very small molecules that includeoxygen ions and peroxides and can be either inorganic or organic and arehighly reactive due to the presence of unpaired valence shell electrons.ROS form as a natural byproduct of the normal metabolism of oxygen andcan also be generated by exogenous sources such as ionizing radiation.Examples of ROS include hydroxide radical, hypochlorite ion, hydrogenperoxide, and superoxide.

The term “reactive nitrogen species” or “RNS” refers to reactivemolecules derived from nitric oxide and superoxide. Examples of RNSinclude peroxynitrite. Reactive nitrogen species act together withreactive oxygen species (ROS) to damage cells, causing nitrosativestress. Therefore, these two species are often collectively referred toas ROS/RNS.

By virtue of their design, the optical sensor conjugates describedherein possess certain advantages and benefits. First, the opticalsensor conjugates are specifically dependent on the ROS/RNS HOCl and/orONOO—, which react with the optical sensor conjugates and cause them torelease the fluorogenic small molecule from the nanoparticle. Second,the optical sensor conjugate design allows for a higher substrate targetconcentration with each nanoparticle delivering approximately 400fluorogenic small molecules to the target. Third, the optical sensorconjugates demonstrate improved pharmacokinetics as compared with thefree (not linked to the nanoparticle) fluorogenic small molecules.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Methods and materials aredescribed herein for use in the present invention; other, suitablemethods and materials known in the art can also be used. The materials,methods, and examples are illustrative only and not intended to belimiting. All publications, patent applications, patents, sequences,database entries, and other references mentioned herein are incorporatedby reference in their entirety. In case of conflict, the presentspecification, including definitions, will control.

Other features and advantages of the invention will be apparent from thefollowing detailed description and figures, and from the claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1a and 1b are schematics of the synthesis of an exemplary opticalsensor conjugate.

FIGS. 1c and 1d are bar graphs depicting the relative fluorescencesignals for a representative fluorescent small molecule and anon-fluorescent small molecule with linker.

FIGS. 1e and 1f are graphs depicting the change in fluorescence signalfor the determination of blood half-life for a free small molecule witha linker and an optical sensor conjugate.

FIGS. 2a and 2b are graphs depicting spectral changes accompanyingactivation of an optical sensor conjugate.

FIGS. 2c and 2d are images showing the color and fluorescence changestriggered by HOCl oxidation and release of the fluorescent smallmolecule.

FIG. 3a is a bar graph depicting the fluorescence response of an opticalsensor conjugate following incubation with a panel of biologicallyrelevant oxidants.

FIG. 3b is a line graph depicting the dependence of a representativeoxazine on increasing HOCl concentration.

FIG. 3c is a line graph showing the effect of chloride ions onactivation of an optical sensor conjugate and release of an oxidizedoxazine.

FIG. 4a is a flow cytometry image showing the ability of an opticalsensor conjugate to detect HOCl generation in a mixture of livingsplenocytes.

FIG. 4b is a graph showing the ability of an optical sensor conjugate todetect HOCl generation in a mixture of living splenocytes by flowcytometry.

FIGS. 5a-h are images of an ex vivo comparison of an optical sensorconjugate or a representative fluorogenic small molecule with theattached linker injected into mouse tissue and the fluorescence signalassociated with a representative oxidized oxazine release, accumulation,and probe washout kinetics.

FIGS. 6a-f are images of in vivo uptake and activation of an MPO opticalsensor conjugate applied to infarcted heart tissue.

FIG. 7a is a bar graph showing fluorescence response of Methylene Blueoptical sensor conjugates with various oxidants.

FIG. 7b is a bar graph showing fluorescence response of Julol. Ox.optical sensor conjugates with various oxidants.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides methods and compositions for detectingROS/RNS in vivo that are the direct result of inflammation. Thesemethods include the use of an optical sensor conjugate that includes afluorogenic small molecule, a nanoparticle, and a linker connecting thefluorogenic small molecule to the nanoparticle (Scheme 1). Thefluorogenic small molecule can be oxidized in vivo by ROS/RNS causingthe small molecule to be released from the conjugatable linker andbecome fluorescent. Thereafter, the fluorescent small molecule can bedetected in vivo by various techniques and systems including flowcytometry, fluorescence reflectance imaging (FRI), fluorescencemolecular tomography (FMT), and microscopic fluorescence imaging.

Structure of Optical Sensor Conjugates

The optical sensor conjugates are comprised of a fluorogenic smallmolecule, a nanoparticle, and a linker connecting the fluorogenic smallmolecule to the nanoparticle.

Fluorogenic Small Molecule

The fluorogenic small molecule can be any low molecular weight organiccompound, i.e., less than about 2000 Daltons, that is “masked” as anon-fluorescent molecule when attached to the linker, but upon reactionwith ROS/RNS, are oxidized and released from the linker as a fluorescentsmall molecule. One such example is an oxazine, e.g., phenoxazine orphenothiazine, which can be oxidized in vivo by either reactive oxygenor reactive nitrogen species, or both, and as a result of the oxidationbecome a fluorescent small molecule. The fluorogenic small molecule canalso contain additional heterocyclic rings, such as but not limited to,piperidine, fused to the phenyl group of the phenoxazine orphenothiazine, carbazine, nile blue, or nile red based fluorophore.

Linker

The term “linker” as used herein refers to a group of atoms, e.g. 3-100atoms, and may be comprised of the atoms or groups, but not limited to,carbon, amino, alkylamino, oxygen, sulfur, sulfoxide, sulfonyl,carbonyl, and imine. The linker is connected to the fluorogenic smallmolecule through an amide bond at a first end of the linker. Inaddition, the linker is connected at a second end to the nanoparticlethrough a covalent bond to a functional group on the nanoparticle. Thecovalent bond is, but is not in any way limited to, an amide bond, anester bond, an ether or amino bond linkage, a triazole moietysynthesized through the reaction of a terminal alkyne and an azide via“click chemistry”.

Various methods are known to attach the linker at the first end to asmall molecule using covalent bonds. Similar methods can also beemployed to attach the linker at the second end to the nanoparticle. Inone example, the linker can be attached to the fluorogenic smallmolecule by forming an amide bond between a carboxyl (or maleimide) onthe linker and the amine located on the fluorogenic small molecule. Inthe same way, the second end of the linker can be attached to thenanoparticle by forming an amide bond between a carboxyl (or maleimide)on the linker and an amine located on the nanoparticle. Reagents thatcan be used include EDC/NHS or SPDP (N-Succinimidyl3-[2-pyridyldithio]-propionate) in both aqueous and organic solvents(such as, but not limited to, dichloromethane, acetonitrile, chloroform,tetrahydrofuran, acetone, formamide, dimethylformamide, pyridine,dioxane, or dimethysulfoxide).

In addition, various methods are known to attach the linker to thenanoparticle. For example, “click chemistry” can be used to attach thelinker to the nanoparticle (see, e.g., the Sigma Aldrich catalog andU.S. Pat. No. 7,375,234, which are both incorporated herein by referencein their entireties). Of the reactions comprising “click” chemistry, oneexample is the Huisgen 1,3-dipolar cycloaddition of alkynes to azides toform 1,4-disubstituted-1,2,3-triazoles. The copper (I)-catalyzedreaction is mild and very efficient, requiring no protecting groups, andrequiring no purification in many cases. The azide and alkyne functionalgroups are generally inert to biological molecules and aqueousenvironments.

The linker may also comprise part of a saturated, unsaturated, oraromatic ring, including polycyclic and heteroaromatic rings wherein theheteroaromatic ring is an aryl group containing from one to fourheteroatoms, N, O, or S. Specific examples include, but are not limitedto, unsaturated alkanes, polyethylene glycols, and dextran polymers.

In addition, the linker must meet the following two criteria. First, thelinker must not interfere with the oxidation of the fluorogenic smallmolecule. Second, the linker can be cleaved from the fluorogenic smallmolecule upon oxidation of the fluorogenic small molecule.

Particle

The optical sensor conjugates include a particle or alternatively apolymer that forms the core of the conjugates. These particles can benanoparticles or microparticles. The term “nanoparticle” refers to aparticle that has a characteristic dimension of less than about 1micrometer, where the characteristic dimension is the largestcross-sectional dimension of a particle. For example, the particle mayhave a characteristic dimension of less than about 500 nm, less thanabout 250 nm, less than about 150 nm, less than about 100 nm, less thanabout 50 nm, less than about 30 nm, less than about 10 nm, or less thanabout 3 nm in some cases. Microparticles with a size of between 1.0 and100 μm can also be employed in the new conjugates. The polymer can havea molecular weight from 2 to 2,000 Kilodaltons.

The particles or polymers useful in the methods and compositionsdescribed herein are made of materials that are (i) biocompatible, i.e.,do not cause a significant adverse reaction in a living animal when usedin pharmaceutically relevant amounts; (ii) feature functional groups towhich the fluorogenic small molecule can be covalently attached; (iii)exhibit low non-specific binding of interactive moieties to theparticles; and (iv) are stable in solution, i.e., the particles orpolymers do not precipitate. Optionally, the particles or polymers caninclude a reporting agent, such as a fluorescent dye. The particles orpolymers can be monodisperse (a single crystal of a material, e.g., ametal, per particle, or have a discrete molecular weight) orpolydisperse (particles with range of different diameters, or polymerswith a range of molecular weights).

A number of particles are known in the art, e.g., organic or inorganicnanoparticles. Liposomes, dendrimers, carbon nanomaterials and polymericmicelles are examples of organic nanoparticles. Inorganic particlesinclude metallic particles, e.g., Au, Ni, Pt and TiO₂ particles. Forexample, magnetic nanoparticles can also be used, e.g., sphericalnanocrystals of 10-20 nm with a Fe²⁺ and/or Fe³⁺ core surrounded bydextran or PEG molecules. In some embodiments, colloidal goldnanoparticles are used, e.g., as described in Qian et al., Nat.Biotechnol. 26: 83-90 (2008); U.S. Pat. Nos. 7,060,121; 7,232,474; andU.S. P.G. Pub. No. 2008/0166706. Suitable multifunctional nanoparticles,and methods for constructing and using multifunctional nanoparticles,are discussed in e.g., Sanvicens and Marco, Trends Biotech., 26:425-433(2008). A number of different polymers are known in the art. These caninclude, but are not limited to dextrans, aminodextrans,carboxymethyldextrans, carboxymethylstarches, polyvinyl alcohols,polyethylene glycols, poly-lysines, or poly-glutamic acids.

The particles or polymers are attached to the linker and thus to thefluorogenic small molecules described herein via a functional group. Insome embodiments, the particles are associated with a polymer thatincludes the functional groups, and also serve to keep the metal oxidesdispersed from each other. The polymer can be a synthetic polymer, suchas, but not limited to, polyethylene glycol or silane, natural polymers,or derivatives of either synthetic or natural polymers or a combinationof these. Useful polymers are hydrophilic. In some embodiments, thepolymer “coating” is not a continuous film around the magnetic metaloxide, but is a “mesh” or “cloud” of extended polymer chains attached toand surrounding the metal oxide. The polymer can comprisepolysaccharides and derivatives, including dextran, pullanan,aminodextran, carboxmethyl dextran, and/or reduced carboxymethyldextran. The metal oxide can be a collection of one or more crystalsthat contact each other, or that are individually entrapped orsurrounded by the polymer.

In some embodiments, the particles are associated with non-polymericfunctional group compositions. Methods are known to synthesizestabilized, functionalized particles without associated polymers, whichare also within the scope of this invention. Such methods for use tomake nanoparticles are described, for example, in Halbreich et al.,Biochimie, 80:379-90, 1998.

In some embodiments, the nanoparticles have an overall size of less thanabout 1-100 nm, e.g., about 25-75 nm, e.g., about 40-60 nm, or about50-60 nm in diameter. The polymer component in some embodiments can bein the form of a coating, e.g., about 5 to 20 nm thick or more. Theoverall size of the nanoparticles is about 15 to 200 nm, e.g., about 20to 100 nm, about 40 to 60 nm; or about 60 nm.

In some embodiments, the fluorogenic small molecule can be conjugated topolymers. For example the fluorogenic small molecule can be conjugatedto poly-lysine or amino dextran, or to larger polymers which can providethe same benefits that the nanoparticles do with regard to in vivopharmacokinetics

In some embodiments, the nanoparticle has a reporter group, such as afluorescent dye attached, e.g., to the functional groups. In oneexample, the nanoparticle can be fluorescence labeled with Alexa Fluor®488 which has an absorption maximum of about 496 nm and an emissionmaximum of 519 nm (green color). It is important that the absorption andemission of the fluorescently labeled nanoparticles not interfere withthe imaging of the fluorescent small molecule.

Synthesis of Particles

There are varieties of ways that the particles can be prepared, but inall methods, the result must be a particle with functional groups thatcan be used to link the nanoparticle to the fluorogenic small molecule.

In all embodiments, the fluorogenic small molecule is attached to theparticle via a linker. The linker is attached to the fluorogenic smallmolecule at a first end and to a functional group on the particle at thesecond end of the linker. There are several methods for placing afunctional group, such as a carboxy or amino, on the particle. Methodsfor synthesizing functionalized, coated particles are discussed infurther detail below.

Carboxy functionalized nanoparticles can be made, for example, accordingto the method of Gorman (see WO 00/61191). Carboxy-functionalizednanoparticles can also be made from polysaccharide coated nanoparticlesby reaction with bromo or chloroacetic acid in strong base to attachcarboxyl groups. In addition, carboxy-functionalized particles can bemade from amino-functionalized nanoparticles by converting amino tocarboxy groups by the use of reagents such as succinic anhydride ormaleic anhydride.

Nanoparticle size can be controlled by adjusting reaction conditions,for example, by varying temperature as described in U.S. Pat. No.5,262,176. Uniform particle size materials can also be made byfractionating the particles using centrifugation, ultrafiltration, orgel filtration, as described, for example in U.S. Pat. No. 5,492,814.

Nanoparticles can also be treated with periodate to form aldehydegroups. The aldehyde-containing nanoparticles can then be reacted with adiamine (e.g., ethylene diamine or hexanediamine), which will form aSchiff base, followed by reduction with sodium borohydride or sodiumcyanoborohydride.

Dextran-coated nanoparticles can also be made and cross-linked, e.g.,with epichlorohydrin. The addition of ammonia will react with epoxygroups to generate amine groups, see Hogemann et al., Bioconjug. Chem.11:941-6 (2000), and Josephson et al., Bioconjug. Chem., 10:186-91(1999).

Carboxy-functionalized nanoparticles can be converted toamino-functionalized magnetic particles by the use of water-solublecarbodiimides and diamines such as ethylene diamine or hexane diamine.

Methods of Detecting Optical Sensors

The compounds and compositions described herein can be used in in vivoimaging methods known in the art, e.g., as described in US 2005/0249668.General principles of fluorescence, optical image acquisition, and imageprocessing can be applied in the practice of the invention. For a reviewof optical imaging techniques, see, e.g., Alfano et al., Ann. NY Acad.Sci., 820:248-270, 1997. Imaging systems typically include three basiccomponents: (1) a near infrared light source, (2) an apparatus forseparating or distinguishing emissions from light used for chromophoreexcitation, and (3) a detection system.

For example, the light source can provide monochromatic (orsubstantially monochromatic) near infrared light. The light source canbe a suitably filtered white light, e.g., bandpass light from abroadband source. For example, light from a 150-watt halogen lamp can bepassed through a suitable bandpass filter commercially available fromOmega Optical (Brattleboro, Vt.). In some embodiments, the light sourceis a laser. See, e.g., Boas et al., Proc. Natl. Acad. Sci. USA91:4887-4891, 1994; Ntziachristos et al., Proc. Natl. Acad. Sci. USA97:2767-2772, 2000; Alexander, J. Clin. Laser Med. Surg. 9:416-418,1991. Information on near infrared lasers for imaging can also be foundon the Internet (e.g., at imds.com) and various other well-knownsources.

A high pass or bandpass filter (e.g., 655 nm) can be used to separateoptical emissions from excitation light. Suitable high pass or bandpassfilters are commercially available from Omega Optical. In someembodiments, a single excitation wavelength can be used to excitemultiple different fluorochromes on a single probe or multiple probes(with different activation sites), and spectral separation with a seriesof bandpass filters, diffraction grating, or other means can be used toindependently read the different activations.

In general, the light detection system can includelight-gathering/image-forming and light-detection/image-recordingcomponents. Although the light-detection system can be a singleintegrated device that incorporates both components, thelight-gathering/image-forming and light-detection/image-recordingcomponents will be discussed separately. However, a recording device maysimply record a single (time varying) scalar intensity instead of animage. For example, a catheter-based recording device can recordinformation from multiple sites simultaneously (i.e., an image), or canreport a scalar signal intensity that is correlated with location byother means (such as a radio-opaque marker at the catheter tip, viewedby fluoroscopy).

A particularly useful light-gathering/image-forming component is anendoscope. Endoscopic devices and techniques that have been used for invivo optical imaging of numerous tissues and organs, includingperitoneum (Gahlen et al., J. Photochem. Photobiol. B 52:131-135,(1999)), ovarian cancer (Major et al., Gynecol. Oncol. 66:122-132,(1997)), colon (Mycek et al., Gastrointest. Endosc. 48:390-394, 1998;Stepp et al., Endoscopy 30:379-386, (1998)) bile ducts (Izuishi et al.,Hepatogastroenterology 46:804-807, (1999)), stomach (Abe et al.,Endoscopy 32:281-286, (2000)), bladder (Kriemair et al., Urol. Int.63:27-31, (1999); Riedl et al., J. Endourol. 13:755-759, (1999)), andbrain (Ward, J. Laser Appl. 10:224-228, (1998)), can be employed in thepractice of the present invention. Other types of light gatheringcomponents useful in the methods described herein are catheter-baseddevices, including fiber optic devices. Such devices are particularlysuitable for intravascular imaging. See, e.g., Tearney et al., Science276:2037-2039, (1997); Boppart et al., Proc. Natl. Acad. Sci. USA94:4256-4261, (1997).

Still other imaging technologies, including phased array technology(Boas et al., Proc. Natl. Acad. Sci. USA 91:48874891, (1994); Chance,Ann. NY Acad. Sci. 838:29-45, (1998)), diffuse optical tomography (Chenget al., Optics Express 3:118-123, (1998); Siegel et al., Optics Express4:287-298, (1999)), intravital microscopy. (Dellian et al., Br. J.Cancer 82:1513-1518, (2000); Monsky et al, Cancer Res. 59:4129-4135,(1999); Fukumura et al., Cell 94:715-725, (1998), fluorescence moleculartomography (FMT), fluorescence reflectance imaging (FRI), and confocalimaging (Korlach et al., Proc. Natl. Acad. Sci. USA 96:8461-8466,(1999); Rajadhyaksha et al., J. Invest. Dermatol. 104:946-952, (1995);Gonzalez et al., J. Med. 30:337-356, (1999)) can be employed in thepractice of the present methods.

Any suitable light-detection/image-recording component, e.g.,charge-coupled device (CCD) systems or photographic film, can be used.The choice of light-detection/image-recording component will depend onfactors including type of light gathering/image forming component beingused. Selecting suitable components, assembling them into a nearinfrared imaging system, and operating the system is within the abilityof a person of ordinary skill in the art.

In addition, the compositions and methods of the present invention canbe used in combination with other imaging compositions and methods. Forexample, the agents of the present invention can be imaged by NIRimaging methods either alone or in combination with other traditionalimaging modalities, such as, X-ray, computed tomography (CT), MRimaging, ultrasound, positron emission tomography (PET), and singlephoton computerized tomography (SPECT). For instance, the methodsdescribed herein can be used in combination with CT or MRI to obtainboth anatomical and molecular information simultaneously, for example,by co-registration of with an image generated by another imagingmodality. The compositions and methods of the present invention can alsobe used in combination with X-ray, CT, PET, ultrasound, SPECT and otheroptical and MR contrast agents.

Uses of Optical Sensor Conjugates

The compounds and compositions described herein are optical sensorconjugates that include a fluorogenic small molecule, an optionallyfluorescent labeled nanoparticle or polymer, and a linker connecting thefluorogenic small molecule to the optionally fluorescence labelednanoparticle. The optical sensor conjugates are useful for detectingand/or monitoring reactive oxygen and/or reactive nitrogen species,which include peroxynitrite (ONOO—) and myeloperoxidase (MPO) mediatedhypochlorous acid (HOCL/OCl—) production. The optical sensor conjugatesare, however, stable toward oxidants such as hydroxyl radical, hydrogenperoxide, and superoxide. The reactive oxygen and reactive nitrogenspecies oxidize the fluorogenic small molecule to release the smallmolecule from the linker and become fluorescent. As a result, thefluorescent small molecule can be detected using flow cytometry,fluorescence reflectance imaging, and microscopic fluorescence imaging.

In Vivo Imaging

The compounds and compositions can be used in in vivo imaging methods.In general, such methods include administering to a subject one or moreoptical sensor conjugates described herein; optionally allowing theoptical sensor conjugates to distribute within the subject; exposing thesubject to light of a wavelength absorbable by at least one fluorophorein the imaging agent; and detecting an optical signal emitted by thefluorophore. The emitted optical signal can be used to construct animage. The image can be a tomographic image. Furthermore, it isunderstood that the methods (or portions thereof) can be repeated atintervals to evaluate the subject over time.

The illuminating and/or detecting steps can be performed using anydevice or apparatus known in the art, e.g., an endoscope, catheter,tomographic system, planar system, hand-held imaging system, or anintraoperative imaging system or microscope.

Before or during these steps, a detection system can be positionedaround or in the vicinity of a subject (for example, an animal or ahuman) to detect signals emitted from the subject. The emitted signalscan be processed to construct an image, for example, a tomographicimage. In addition, the processed signals can be displayed as imageseither alone or as combined images.

Information provided by such in vivo imaging, for example, the presence,absence, or level of emitted signal, can be used to detect and/ormonitor tissue damage, inflammation, and/or disease in the subject.Examples of causes of tissue damage include, without limitation,Alzheimer's disease, atherosclerosis, cancer, stroke, inflammatory boweldisease, and organ transplant. In addition, in vivo imaging can be usedto assess the effect of a compound or therapy by using the opticalsensor conjugate, wherein the subject is imaged prior to and aftertreatment with the compound or therapy, and the correspondingsignal/images are compared.

The methods and compositions described herein can be used to help aphysician or surgeon to identify and characterize areas of disease, suchas cancers and atherosclerosis, and to distinguish between dead or dyingtissue after suffering a heart attack or stroke.

The methods and compositions described herein can also be used in thedetection, characterization, and/or determination of the localization ofa disease, especially early disease, the severity of a disease or adisease-associated condition, the staging of a disease, and/ormonitoring a disease. The presence, absence, or level of an emittedsignal can be indicative of a disease state.

The methods and compositions disclosed herein can also be used tomonitor and/or guide various therapeutic interventions, such as surgicalprocedures, and monitoring drug therapy, including cell based therapies.The methods can also be used in prognosis of a disease or diseasecondition.

With respect to each of the foregoing, examples of such disease ordisease conditions that can be detected and/or monitored (before, duringor after therapy) include inflammation (for example, inflammation causedby arthritis, for example, rheumatoid arthritis), cancer (for example,colorectal, ovarian, lung, breast, prostate, cervical, testicular, skin,brain, gastrointestinal, pancreatic, liver, kidney, bladder, stomach,leukemia, mouth, esophageal, bone), cardiovascular disease (for example,atherosclerosis and inflammatory conditions of blood vessels, ischemia,hypertension, stroke, myocardial infarction, thrombosis, disseminatedintravascular coagulation), dermatologic disease (for example, Kaposi'sSarcoma, psoriasis, allergic dermatitis), ophthalmic disease (forexample, macular degeneration, diabetic retinopathy), infectious disease(for example, bacterial, viral, fungal and parasitic infections,including Acquired Immunodeficiency Syndrome, Malaria, Chagas Disease,Schistosomiasis), immunologic disease (for example, an autoimmunedisorder, lymphoma, multiple sclerosis, rheumatoid arthritis, diabetesmellitus, lupus erythematosis, myasthenia gravis, Graves disease),central nervous system disease (for example, a neurodegenerativedisease, such as Parkinson's disease or Alzheimer's disease,Huntington's Disease, amyotrophic lateral sclerosis, prion disease),inherited diseases, metabolic diseases, environmental diseases (forexample, lead, mercury and radioactive poisoning, skin cancer),bone-related disease (for example, osteoporosis, primary and metastaticbone tumors, osteoarthritis), neurodegenerative disease, andsurgery-related complications (such as graft rejection, organ rejection,alterations in wound healing, fibrosis or other complications related tosurgical implants).

The methods and compositions described herein, therefore, can be used,for example, to determine the presence and/or localization of tumorcells, the presence and/or localization of inflammation, including thepresence of activated macrophages, for instance in atherosclerosis orarthritis, the presence and localization of vascular disease includingareas at risk for acute occlusion (i.e., vulnerable plaques) in coronaryand peripheral arteries, regions of expanding aneurysms, unstable plaquein carotid arteries, and ischemic areas. The methods and compositionsdescribed herein, can also be used in identification and evaluation ofcell death, injury, apoptosis, necrosis, hypoxia and angiogenesis. Themethods and compositions can also be used for drug delivery and tomonitor drug delivery, especially when drugs or drug-like molecules arechemically attached to the imaging agents.

Compositions

The optical sensor conjugates described herein can be provided dry ordissolved in a carrier or vehicle, e.g., pharmaceutically acceptablecarriers and vehicles. Useful carriers and vehicles include, but are notlimited to, buffer substances such as phosphate, glycine, sorbic acid,potassium sorbate, tris(hydroxymethyl)amino methane (“TRIS”), partialglyceride mixtures of fatty acids, water, salts or electrolytes,disodium hydrogen phosphate, potassium hydrogen phosphate, and sodiumchloride.

The optical sensor conjugates can be administered in the form of asterile injectable preparation. The possible vehicles or solvents thatcan be used to make injectable preparations include water, Ringer'ssolution, and isotonic sodium chloride solution, and 5% D-glucosesolution (D5W). In addition, oils such as mono- or di-glycerides andfatty acids such as oleic acid and its derivatives can be used. Thecompounds and compositions can be administered orally, parenterally, byinhalation, topically, rectally, nasally, buccally, vaginally, or via animplanted reservoir. The term “parenteral administration” includesintravenous, intramuscular, intra-articular, intrasynovial,intrasternal, intrathecal, intraperitoneal, intracisternal,intrahepatic, intralesional, and intracranial injection or infusiontechniques. The optical sensor conjugates can also be administered viacatheters or through a needle to any tissue.

Dosing of the optical sensor conjugate will depend on a number offactors including the sensitivity of the detection system used, as wellas a number of subject-related variables, including animal species, age,body weight, mode of administration, sex, diet, time of administration,and rate of excretion.

Prior to use of the invention or any pharmaceutical composition of theinvention, the subject can be treated with an agent or regimen toenhance the imaging process. For example, a subject can be put on aspecial diet prior to imaging to reduce any auto-fluorescence orinterference from ingested food, such as a low pheophorbide diet toreduce interference from fluorescent pheophorbides that are derived fromsome foods, such as green vegetables. Alternatively, a cleansing regimencan be used prior to imaging, such as those cleansing regimens that areused prior to colonoscopies and include use of agents such as Visiciol™.The subject (patient or animal) can also be treated with pharmacologicalmodifiers to improve image quality. For example, using low doseenzymatic inhibitors to decrease background signal relative to targetsignal (secondary to proportionally lowering enzymatic activity ofalready low-enzymatic activity normal tissues to a greater extent thanenzymatically-active pathological tissues) can improve thetarget-to-background ratio during disease screening.

EXAMPLES

The invention is further described in the following examples, which donot limit the scope of the invention described in the claims.

Example 1 Synthesis of an Optical Sensor Conjugate Employing an Oxazine

Oxazine, as its perchlorate salt, was purchased from Acros Organics USA(Morris Plains, N.J.) and used as received. Alexafluor® 488 (AF488)succinimidyl ester was purchased from Molecular Probes. All otherchemicals and solvents, unless noted, were purchased from Sigma-Aldrichor Fisher Scientific. Preparative high performance liquid chromatography(HPLC) was performed on a Varian 210 instrument equipped with a 335diode array detector and a Varian Pursuit XRs 10 C18 250×21.2 mm columnat a flow rate of 20 mL/min. All ¹H (400 MHz) and ¹³C NMR (100 MHz)nuclear magnetic resonance (NMR) spectra were acquired on a BrukerDPX-400 spectrometer at ambient temperature and were referenced totetramethylsilane as an internal standard. Absorption spectra werecollected on a Varian Cary 50-Bio UV/visible spectrophotometer.

For determination of the extinction coefficient of the fluorescent smallmolecule, fresh stock solutions of the fluorescent small molecule wereprepared for each trial by dissolution of 2-4 mg portions of the dye,weighed on a Mettler AT201 analytical balance with an error of ±0.01 mg,in PBS, pH 7.4, using a 10 mL volumetric flask. Standard deviations forthe extinction coefficient measurements, performed in triplicate, were5% or less. Fluorescence data were collected on a Varian Cary Eclipsefluorescence spectrophotometer. High-resolution electrospray ionization(ESI) mass spectra were collected on a Bruker Daltonics APEXII 3 TFourier transform mass spectrometer in the Department of ChemistryInstrumentation Facility at the Massachusetts Institute of Technology.

Synthesis of sodium5-(3,7-bis(diethylamino)-10H-phenoxazin-10-yl)-5-oxopentanoate(intermediate 1). To oxazine perchlorate (1) (42.4 mg, 0.1 mmol) andglutaric anhydride (114 mg, 1 mmol) in chlorobenzene (0.5 mL) was addedtriethylamine (279 μL, 2 mmol). The dark blue mixture was heated in asealed thick-walled pressure tube at 150° C. for 30 min. After coolingthe now brown reaction mixture was concentrated by rotary evaporationand the crude product was re-dissolved in DMF (2 mL).

Preparative HPLC of the crude reaction using a gradient from 0 to 50%buffer B over 30 min afforded the purified intermediate as its freeacid. Buffer A consists of water with 0.1% trifluoroacetic acid (TFA)and buffer B is acetonitrile with 10% water and 0.1% TFA. The productwas concentrated to ˜5 mL by rotary evaporation. When dried, the freeacid of the intermediate is very hygroscopic. To this hygroscopic form,the 5 mL acidic solution containing the free acid was neutralized with0.1 M bicarbonate buffer, pH 7.4. After neutralization, the cloudysuspension was loaded onto a 10 g reverse phase C18 silica-desaltingcolumn (Waters Corp.). The excess inorganic salts were removed bywashing with 50 mL of deionized water, and pure intermediate 1 (30.0 mg,65%) as its sodium salt was isolated by elution with a mixture ofacetonitrile containing 25% water. The purity of the product wasverified to be >97% by analytical HPLC.

¹NMR (400 MHz, CD₃OD): δ 7.24 (d, 2H, J=8.4 Hz), 6.42-6.38 (m, 4H), 3.32(quartet, 8H, J=7.1 Hz), 2.63 (t, 2H, J=7.5 Hz), 2.17 (t, 2H, J=7.5 Hz),1.88 (pentet, 2H, J=7.4 Hz), 1.11 (t, 12H, J=7.0 Hz). ¹³C NMR (400 MHz,CD₃OD): δ 181.8, 175.3, 154.6, 149.2, 127.4, 120.1, 108.4, 101.5, 46.4,35.5, 34.5, 24.1, 13.7. HRMS-ESI [M−H]⁻ m/z calcd. for [C₂₅H₃₂N₃O₄]⁻438.2398, found 428.2398.

Synthesis of the ROS/RNS nanoparticle conjugate. Alexa Fluor 488 labeledCLIO nanoparticles (hydrodynamic diameter 41 nm as determined by dynamiclight scattering) were prepared according to published procedures (Kochet al. Bioconjug Chem 14, 1115-21, (2003). To a solution of CLIO-AF488(3 mg Fe) in 2.7 mL of PBS, pH 7.4 was added intermediate 1 (3 mg, 6.5μmol) dissolved in 300 μL of DMSO andN-(3-Dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride (EDAC, 30mg, 0.16 mmol). This solution was allowed to stir in overnight (dark,room temperature). Following incubation, the labeled conjugate waspurified by size exclusion chromatography (Sephadex G-50 resin) elutingwith PBS, pH 7.4. The purified nanoparticle solution was concentrated to1 mL by centrifugal filtration with an Amicon Ultra 3,000 MW cutoffcentrifugal filter. The concentration of this stock solution (2.25 mgFe/mL) was determined by comparing the absorbance at 400 nm (awavelength at which AF488 and intermediate 1 do not absorb) to a dilutedCLIO-AF488 stock solution of known concentration.

The number of fluorogenic small molecules with linker per nanoparticle,which have no absorption in the visible spectrum, was determined byactivation of a dilute 10 nM solution of the final optical sensorconjugate with excess NaOCl or peroxynitrite. The concentration of thereleased oxazine fluorophore was then measured using its extinctioncoefficient in PBS (ε=107,000 M-1 cm-1). Using this procedure, thenumber of ROS activatable oxazine groups per nanoparticle is calculatedto be ˜400.

The optical sensor conjugate was synthesized by the reaction scheme inFIG. 1a-b and is based on the HOCl/ONOO⁻ dependent release of the smallmolecule from the parent nanoparticle. The linker was engineered to beattached to the small molecule (FIG. 1a ) that is non-fluorescent (FIG.1c ), which was used for attachment to the Alexa Flour 488-modifiedmagnetic nanoparticle (FIG. 1b ). The nanoparticle by itself (without asmall molecule) has been studied extensively (Josephson et al. BioconjugChem, 10:186-91 (1999)) and has been used in humans (Harisinghani, etal. Neoplasia, 9:1160-5 (2007)). This sensor design results in a highsubstrate target concentration with each nanoparticle containing ˜400activatable oxazine functionalities. Activation of the optical sensorconjugate by HOCl generated from the MPO/H₂O₂/Cl⁻ system results inrelease of the fluorescent small molecule from the nanoparticle scaffoldand restoration of its fluorescent properties (FIG. 1d ), whileretaining the covalent Alexa Fluor 488 labels (FIG. 1b ).

Example 2 Optical Sensor Conjugate Blood Half-Life

In vivo pharmacokinetics of the small molecule alone and the opticalsensor conjugate were determined in groups of mice. Each group consistedof 5 mice (C57/B6, Jackson Labs) that were injected either with thesmall molecule, oxazine (50 nmol), or the nanoparticle, optical sensorconjugate (8-10 mg/kg body weight) while anesthetized (isoflurane 2-3%v/v+2 L/min O₂). Retro-orbital bleeds were taken at various times andimmediately transferred to a tube containing anticoagulant heparin. Theblood samples were then imaged on the Bonsai fluorescence reflectanceimaging system (Siemens) using the Cy 5 channel (l_(exc)=620-650 nm withl_(em) 680-710 nm) for oxazine and GFP channel (l_(exc)=450-480 nm withl_(em) 500-530 nm) for the AF488-labeled optical sensor conjugate due tothe quenched nature of the fluorogenic small molecule attached to thenanoparticles. Fluorescence measurements were corrected for controlbackground levels (blood from un-injected control mice) and expressed aspercent injected dose.

The attachment of the small molecule to the nanoparticle significantlyincreases the blood half-life of the circulating conjugate from ˜40minutes to over 9 hours (FIGS. 1e-f ), as compared to the free smallmolecule, making it suitable for animal imaging. The oxidation of thesensor causes absorbance spectral changes as shown in FIG. 2a , markedby the generation the free oxazine with l_(max) of 655 nm (ε=107,000 M⁻¹cm⁻¹) and a dramatic reaction color change from orange to green (FIG. 2c). Activation of the optical sensor conjugate and release of thefluorescent small molecule after exposure to excess NaOCl generatesa >500-fold increase in fluorescence emission at l_(max) of 672 nm(l_(exc)=620 nm, FIG. 2b ) and release of the fluorescent dye (FIG. 2d).

The specificity of the optical sensor conjugate towards activation by apanel of ROS species was determined, with only HOCl and ONOO⁻ able tocause oxazine release (FIG. 3a ). Importantly, no activation wasobserved by H₂O₂, OH—, NO/O₂, and O₂. Both HOCl/OCl⁻ and ONOO⁻ are knownto be induced in ischemia/reperfusion injury (Yasmin et al. CardiovascRes 33:422-32 (1997)). The optical sensor conjugate activation is linearover μM concentration range of NaOCl (FIG. 3b ). The rate of HOClproduction by stimulated neutrophils is ˜0.1 fM HOCl/sec (Aratani et al.Infect Immun 67:1828-36 (1999)), assuming 10⁶ neutrophils in a mouseinfarct this would represent 0.1 nM HOCl/sec. The actual concentrationof HOCl produced by myeloid cells in vivo in response to injury isunclear due to confounding factors such as in vivo cellular half-life ofthe ROS species, variability of tissue response to ischemic trauma, andrate of extracellular HOCl diffusion. Progress curves for MPO-dependentactivation of the optical sensor conjugate in the presence and absenceof Cl⁻ ions indicate activation of the optical sensor conjugate isstringently dependent on the formation of HOCl and not simply on thecontent of OH— radicals or H₂O₂ (FIG. 3c ).

Example 3 Selectivity of the Optical Sensor Conjugate for ROS Species

All in vitro activation experiments were performed in triplicate usingthe optical sensor conjugate in PBS (10 mM phosphate, 2.7 mM KCl, 137 mMNaCl, pH 7.4) at a 5 nM concentration (2 μM with respect to activatableoxazine). Selectivity of the optical sensor conjugate was determined byscreening against a panel of biologically relevant oxidants. Theintegrated fluorescence response from 650 to 850 nm (λ_(ex)=620 nm) ofthe optical sensor conjugate was measured 30 min after treatment withthe appropriate ROS or RNS at room temperature. The final oxidantconcentration is 25 μM except for H₂O₂ (250 μM) and for the Fentonreagents (Fe(ClO₄)₂: 50 μM and H₂O₂: 250 μM) to generate HO. Otheroxidant sources include: Sodium hypochlorite (Sigma-Aldrich),peroxynitrite in 4.7% aqueous NaOH (EMD Biosciences), NOC-9 nitric oxidedonor (EMD Biosciences), potassium superoxide (Strem Chemical), and2,2′-(azobis(2-amidinopropane)dihydrochloride alkylperoxy radical source(EMD Biosciences). Photo-oxidation was investigated by placing theoptical sensor conjugate under a fluorescent lamp for 2 h. For allROS/RNS assays the final buffer pH was 7.4±0.1.

To determine whether the optical sensor conjugate is capable ofspecifically monitoring MPO-dependent reactions in living cells, flowcytometry studies were performed using mouse CD11b enriched leukocytes.The flow cytometry gating strategy exploited here (FIG. 4a ), namelyB220/CD90/CD49b/NK1.1./Ly6G/Ter119 versus CD11b, has been usedpreviously (Nahrendorf et al. Circulation 117: 379-87 (2008)). The invivo optical sensor conjugate activation indicates that the fluorescencesignal is MPO-dependent and can be blocked by peroxidase inhibition(FIG. 4b ). Populations of neutrophils were confirmed by cytospinmorphologic analysis after H&E staining (insets, FIG. 4b ). Furthermore,HOCl generation is dominant with respect to ONOO— production inneutrophils as indicated by the ˜95% reduction in MPO/ROS optical sensorconjugate activation caused by inhibiting MPO function. Thecompatibility of the small molecule fluorescence wavelengths withstandard APC filter sets will also enable future flow cytometry studiesaimed at screening for suppressors of the MPO/H₂O₂/Cl⁻ system inmonocytes/macrophages and neutrophils.

Example 4 Isolation of Mouse Neutrophils

Flow sorting of murine neutrophils and monocyte/macrophages wasperformed as previously described (Nahrendorf et al. J Exp Med 204:3037-47 (2007)). Briefly, spleens from euthanized mice were removed,triturated in DPBS (Lonza Inc.) at 4° C. with the end of a 3 mL syringeand filtered through 40 μm nylon mesh (BD Bioscience). Cell suspensionswere centrifuged at 1500 rpm for 10 min at 4° C. Red blood cells werelysed with a hypo-osmolar ACK lysis buffer and the splenocytes werewashed and resuspended in DPBS supplemented with 0.5% bovine serumalbumin (BSA) and 1% fetal calf serum (FCS). Splenocytes were labeledwith the following antibodies CD90-PE/53-2.1, B220-PE/RA3-6B2,CD49b-PE/DX5, NK1.1.-PE/PK136, Ly6G-PE/1A8, Ter119-PE/Ter119,CD11b-APC-Cy7/M1/70 (Antigen-Fluorochrome/Clone, all from BD Bioscience)for 30 min. at 4° C. Labeled splenocytes were FACS sorted on a FACS Ariainstrument (BD Bioscience) according to the expression of the myeloidmarker CD11b. This CD11b⁺ enriched cell population yielded a purityof >98% myeloid cells consisting of primarily neutrophils. Formorphologic characterization, sorted cells were spun, resuspended in 300μL DPBS and prepared on glass slides by cytocentrifugation (Shandon,Inc.) at 10 G for 5 min and stained with HEMA-3 (Thermo FisherScientific). These CD11b⁺ enriched cells were then used in Example 5 forin vivo studies.

Example 5 In Vivo Activation of Optical Sensor Conjugates

For intracellular probe activation, 100 K of CD11b⁺-enriched cells wereincubated with the optical sensor conjugate at a final concentration of20 μM for 2 h at 4° C. Control cells were incubated in DPBS supplementedwith 0.5% BSA and 1% FCS. For inhibitor studies, 4-aminobenzoichydrazide was added at a final concentration of 10 μM. After theincubation period, cells were washed and analyzed by flow cytometry on aLSRII Cytometer (BD Bioscience) after appropriate compensations.Neutrophils were defined as CD11b^(hi)Ly6G^(hi), monocyte/macrophages asCD11b^(hi)(B220/CD90/CD49b/NK1.1./Ly6G/Ter119)^(lo). Activation of theoptical sensor conjugate was quantified in the allophycocyanine (APC)channel. Mean fluorescence intensities are shown in FIG. 4 b.

To investigate the utility of the optical sensor conjugate in vivo, wedetermined the ability of the sensor to monitor ischemia andinflammation as a result of permanent coronary ligation in a mouse modelof myocardial infarction. For all experiments, the agents (opticalsensor conjugate or fluorogenic small molecule with linker) wereinjected intravenously into the tail-vein of the mice 12-14 hours afterMI and imaged 24 hours later when neutrophil recruitment to the infarctis high. Macroscopic analysis by fluorescence reflectance imaging ofcoronal sections of the infarcted mouse hearts indicated that theoptical sensor conjugate localized to the infarcted zone, whereas thefluorescence from the small molecule probe had washed out of the infarctand into the myocardium (FIG. 5a-h ).

Microscopic analysis of tissue sections from infarcted hearts confirmsthat small molecule fluorescence coincides with areas rich in MPO andneutrophils, which are the dominant cells recruited early afterinfarction. H&E, Ca11b, neutrophil and MPO staining show correlationwith the oxazine signal and nanoparticle cellular localization (FIG.6a-f ). The biocompatibility of the optical sensor conjugate and itsfavorable pharmacokinetics will enable non-invasive fluorescencemolecular tomography and other optical imaging studies to be performedto identify areas of inflammation caused by chronic disease or ischemicinjury.

Example 6 Tail Vein Administration of the Optical Sensor Conjugate andDeposition/Activation in Ischemic Mouse Heart Tissue after MyocardialInfarction (MI)

Myocardial infarcts were induced in 12 mice (C57/B6, Jackson Labs) bycoronary ligation during inhalation anesthesia (isoflurane 2-3% v/v+2L/min O₂) as previously described (Nahrendorf et al. Circulation,113:1196-202 (2006)), with 6 mice serving as controls which did notreceive coronary ligation. Anesthesized mice were intubated and athoracotomy was performed in the 4^(th) left intercostal space tovisualize the left ventricle where the left coronary artery was ligated.The chest wall was closed and the mouse recovered after extubation.Approximately 12 hours following the induction of the infarct, theoptical sensor conjugate was injected via tail vein as a 100-150 μLbolus (15 mg/kg). The hearts were harvested 36 hours after MI.

Hearts were excised and rinsed in PBS and cut into myocardial rings of1-mm thickness and either immediately stained with 2-3-5-triphenyltetrazolium chloride (TTC) (Nahrendorf et al. Circulation, 113:1196-202(2006)) to highlight the infarcted tissue or used for histologicalanalysis. Fluorescence reflectance imaging of side-by-side myocardialrings of controls and injected hearts was performed in the GFP channel(l_(exc)=450-480 nm with l_(em) 500-530 nm) and Cy5 channel(l_(exc)=620-650 nm with l_(em)=680-710 nm) using an Olympus OV-100system (Olympus, Center Valley, Pa.). For histology and fluorescencemicroscopy, the tissue was embedded in OCT medium (Sakura Finetek,Torrance, Calif.). Serial 6-μm-thick sections were collected in themidventricular level and used for immunohistochemical staining for CD11b(Abcam, Cambridge, Mass.), neutrophils (NIMP-R14, Abcam, Cambridge,Mass.) and MPO (NeoMarkers, Freemont, Calif.). The reaction wasvisualized as a 3-step staining procedure using biotinylated secondaryantibodies (BA4001, Vector Laboratories, Burlingame, Calif.) and the AECSubstrate Kit (Vector Laboratories). Sakura Finetek Torrance, Calif.).Multichannel fluorescence microscopy was used to assess probelocalization in the tissue sections with an upright epifluorescencemicroscope (Eclipse 80i, Nikon Instruments, Melville, N.Y.).Fluorescence microscopy images were obtained using at a green/GFP filter(Q505LP bandpass, l_(exc)=480±20 nm with l_(em)=535±25 nm) and a far-redfilter (Q680LP bandpass, l_(exc)=650±23 nm with l_(em)=710±25 nm). Theseresults indicate that the conjugates can be used to clearly imagemyocardial infarction in living animals.

Example 7 Synthesis of an Optical Sensor Conjugate Employing MethyleneBlue

Synthesis of sodium5-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-5-oxopentanoate (MBintermediate, step (i))

Methylene blue (MB, 3,7-bis(Dimethylamino)phenazathionium chloride, 0.1mmol) was dissolved in acetonitrile (1 mL). Glutaric anhydride (1.0mmol) and sodium hydrosulfite (0.5 mmol) were then added to thesolution. The reaction mixture was then subjected to microwaveirradiation with the following settings: T=150° C., t=20 minutes,power=300 Watts, Pmax=off, using a CEM Discover Microwave Model 908005.The insoluble sodium hydrosulfite was then removed by centrifugation,washing 2 times with acetonitrile, decanting off the green-coloredsupernatant. The crude product was then purified by preparative HPLCusing a gradient from 0 to 90% acetonitrile containing 0.1%trifluoroacetic acid over 30 minutes. The product was concentrated byrotary evaporation to approximately ⅓ the original volume and the acidicsolution was neutralized with 0.1 M sodium bicarbonate buffer, pH 8.0.The cloudy suspension was then loaded onto a reverse phase C18silica-desalting column, which was then flushed with deionized water toremove excess salts before eluting the purified the MB intermediate asits sodium salt using a 1:3 mixture of acetonitrile (27% yield).

¹NMR (500 MHz, CD₃OD) δ 7.31 (d, 2H, J=7.5 Hz), 6.81 (d, 2H, J=3.0 Hz),6.72 (dd, 2H, J=2.5, 9.0 Hz), 2.95 (s, 12H), 2.49 (broad s, 2H), 2.25(t, 2H, J=7.0 Hz), 1.82 (pentet, 2H, J=7.5 Hz) ESI-MS [M+H]⁺ (m/z)calcd. for C21H26N3O3S⁺; 400.17 m/z, found 400.2 m/z.

Synthesis of 2,5-dioxopyrrolidin-1-yl5-(3,7-bis(dimethylamino)-10H-phenothiazin-10-yl)-5-oxopentanoate (MBintermediate SE, step (ii))

N,N′-disuccinimidyl carbonate (70 μmol) and diisopropylethylamine (70μmol) were added to a solution of the MB intermediate (17 μmol) in 1 mLof DMSO. This reaction mixture was allowed to stir at room temperaturefor 1 hour or until completion as determined by consumption of thestarting material using LCMS. The title product was identified by a newpeak in the LC trace slightly more non-polar than the starting materialwith a corresponding mass of 497.3 (ESI MS [M+H]⁺ (m/z) calcd. forC₂₅H₂₉N₄O₅S⁺; 497.18).

Synthesis of the MB Optical Sensor Conjugate

CLIO nanoparticles (5 mg Fe) were added directly to the crude MBintermediate SE reaction mixture from above after completion (17 μmolproduct if quantitative), followed by the addition of 1 mL of DMSO and 3mL sodium phosphate buffer (10 mM, pH 8.0). This solution was thenallowed to stir overnight in the dark at room temperature. The MBintermedate conjugated nanoparticles were then purified by sizeexclusion chromatography (Sephadex G-25 resin) eluting with PBS, pH 7.4.This purified nanoparticle solution was then concentrated using anAmicon Ultra 5K MWCO centrifugal filter. The nanoparticle concentrationwas determined by comparing the absorbance at 400 nm to a knownunmodified CLIO dilution. The number of quenched methylene bluefluorophores per nanoparticle was then determined by release of thefluorophore by activation with excess HOCl . The extinction coefficientat 664 nm (ε=74,000 M⁻¹cm⁻¹ in water) was used to calculate the amountof fluorophore released. This yielded approximately 70 activatablemethylene blue groups per nanoparticle.

Example 8 Synthesis of an Optical Sensor Conjugate Employing8-Hydroxyjulolidine

Synthesis of2,3,6,7,12,13,16,17-Octahydro-1H,5H,11H,15H-diquinolizino[1,9-bc:1′,9′-hi]phenoxazin-4-iumchloride (Julol. Ox., step (I))

Sodium nitrite (4 mmol) dissolved in water was added dropwise to8-hydroxyjulolidine (8 mmol) stirring in acetic acid on ice. The nowbrownish solution was then stirred for 30 minutes before heating to 65°C. for 4 hours. The acetic acid was then removed from the dark bluesolution by rotary evaporation and the title product as itstrifluoroacetate salt was purified by HPLC using a gradient from 25 to90% acetonitrile containing 0.1% trifluoroacetic acid over 30 min. HPLCfractions containing the product were concentrated by rotary evaporationand loaded onto a reverse phase C18 column and washed with 250 mL 0.1%HCl to exchange the trifluoroacetate counteranion for chloride. Thechloride salt of the title compound was then eluted with 50%acetonitrile in water and lyophilized to give a dark blue powder (25%yield). ESI-MS [M]⁺ (m/z) calcd. for C₂₄H₂₆N₃O⁺; 372.21, found 372.3.

Synthesis of sodium5-(2,3,6,7,12,13,16,17-octahydro-1H-diquinolizino[1,9-bc:1′,9′-hi]phenoxazin-9(5H,11H,15H)-yl)-5-oxopentanoate(Julol. Ox. intermediate, step (II))

Julol. Ox. (0.1 mmol) and glutaric anhydride (1 mmol) were dissolved in2 mL dichloroethane in a thick-walled pressure tube. Triethylamine (2mmol) was then added before sealing the tube and heating to 125° C. forapproximately 15 minutes or until the visible blue color disappears andthe solution becomes dark brown. The solvent was removed by rotaryevaporation and the title compound was purified by HPLC afterredissolving the brown solid in DMF using a gradient from 0 to 90%acetonitrile containing 0.1% trifluoroacetic acid over 30 minutes. Theproduct fractions were concentrated by rotary evaporation toapproximately ⅓ the original volume and the acidic solution wasneutralized with 0.1 M sodium bicarbonate buffer, pH 8.0. The cloudysuspension was then loaded onto a reverse phase C18 silica-desaltingcolumn, which was then flushed with deionized water to remove excesssalts before eluting the purified Julol. Ox. intermediate as its sodiumsalt using a 1:1 mixture of acetonitrile:water that was lyophilized todryness (42% yield). ESI MS [M+H]⁺ (m/z) calcd. for C₂₉H₃₄N₃O₄ ⁺;488.25, found 488.3.

Synthesis of 2,5-dioxopyrrolidin-1-yl5-(2,3,6,7,12,13,16,17-octahydro-1H-diquinolizino[1,9-bc:1′,9′-hi]phenoxazin-9(5H,11H,15H)-yl)-5-oxopentanoate(Julol. Ox. intermediate SE, step (III))

Julol. Ox. intermediate (0.04 mmol) and N,N′-disuccinimidyl carbonate(0.16 mmol) were dissolved in 2 mL of DMSO. Diisoproplyethylamine (16mmol) was then added and the solution was allowed to stir at roomtemperature for 1 hour or until completion as determined by completeconsumption of the starting material using LCMS. The title product wasidentified by a new peak in the LC trace slightly more non-polar thanthe starting material with a corresponding mass of 585.4 (ESI MS [M+H]⁺(m/z) calcd. for C₃₃H₃₇N₄O₆ ⁺; 585.27).

Nanoparticle Conjugation of Julol. Ox. Intermediate SE

CLIO nanoparticles (5 mg Fe) were added directly to the crude Julol. Ox.intermediate SE reaction mixture from above (40 μmol product ifquantitative), followed by the addition of 2.5 mL of DMSO and 1.8 mLPBS, pH 7.4. This solution was then allowed to mix overnight in the darkat room temperature. The Julol. Ox. intermediate conjugatednanoparticles were then purified by size exclusion chromatography(Sephadex G-25 resin) eluting with PBS, pH 7.4. This purifiednanoparticle solution was then concentrated using an Amicon Ultra 5KMWCO centrifugal filter. The nanoparticle concentration was determinedby comparing the absorbance at 400 nm to a known unmodified CLIOdilution. An estimated number of quenched Julol. Ox. per nanoparticlewas then determined by release of the fluorophore by activation withexcess HOCl. The approximate extinction coefficient at 675 nm (ε=100,000M⁻¹cm⁻¹ in PBS) was used to calculate the amount of fluorophorereleased. This yielded 40 activatable Julol. Ox. groups pernanoparticle.

Example 9 Oxidant Activation Assays of MB Optical Sensor Conjugates andJulol. Ox. Optical Sensor Conjugates

The quenched dye functionalized nanoparticles were then subjected toscreening with a panel of biologically relevant reactive oxygen andreactive nitrogen species (ROS/RNS). Assays were performed in triplicatein PBS buffer containing 10 mM phosphate, 2.7 mM KCl, 137 mM NaCl pH7.4. The amount of nanoparticle used was such that the finalconcentration of fluorophore would be 2 μM upon complete activation. Theintegrated fluorescence response from 665-850 nm (λ_(ex)=640 nm) formethylene blue and from 675-850 nm (λ_(ex)=650 nm) for Julol. Ox. wasmeasured after treatment with the individual ROS/RNS for 30 minutes' atroom temperature. The final oxidant concentration was 25 μM with theexception of H₂O₂ (250 μM) and the Fenton reagents (Fe(ClO₄)₂: 50 μM andH₂O₂: 250 μM) used to generate HO. Photo-oxidation was investigated byplacing the functionalized nanoparticles under fluorescent light for 2hours. The results of these assays can be seen in FIGS. 7a and 7b . Boththe MB and Julol. Ox. optical sensor conjugates showed specificitytoward the ROS/RNS, HOCl and ONOO—.

Other Embodiments

It is to be understood that while the invention has been described inconjunction with the detailed description thereof, the foregoingdescription is intended to illustrate and not limit the scope of theinvention, which is defined by the scope of the appended claims. Otheraspects, advantages, and modifications are within the scope of thefollowing claims.

What is claimed is:
 1. A method of imaging reactive oxygen species,reactive nitrogen species, or both in a subject, the method comprising:administering to the subject an optical sensor conjugate comprising afluorogenic small molecule, a particle or polymer, and a linker thatconnects the small molecule to the particle or polymer, for a timesufficient for the small molecule to react with the reactive oxygen orreactive nitrogen, or both, thereby releasing the small molecule fromthe optical sensor conjugate and becoming fluorescent; and obtaining afluorescence image of the small molecule in the subject, thereby imagingthe subject, wherein the small molecule with the linker comprises acompound of Formula I:

wherein: Z is the linker and comprises C₃₋₂₀ alkyl, wherein any of thecarbons in C₃₋₂₀ alkyl can be replaced with —C(O)—, C(O)O—,—C(O)NR^(A)—, —C(NH)—, oxygen, sulfur, —SO₂—, —NR^(A)SO₂— —NR^(A)—; X isselected from

 N₃, C(O)Cl, and C(O)OR^(A); Y is selected from O, S, Se, Te, N(R^(A)),and C(Me)₂; R¹ is selected from H, C₁₋₆ alkyl, OR^(A), and NR^(C)R^(D),wherein said C₁₋₆ alkyl is optionally substituted by OR^(A) orNR^(C)R^(D); or 2 or 3 R¹ adjacent to each other and together with the Catoms to which they are attached form 1or 2 heterocycloalkyl, optionallysubstituted by 1, 2, 3, or 4 substituents independently selected from H,OH, C₁₋₆ alkyl, OR^(A), C(O)R^(B), C(O)NR^(C)R^(D), and C(O)OR^(A);R^(A), R^(B), R^(C) and R^(D) are independently selected from H, C₁₋₆alkyl, and succinimidyl; and m is 1, 2, 3, or
 4. 2. The method of claim1, wherein X is C(O)OR^(A) and Y is O.
 3. The method of claim 1, whereinR¹ is NR^(C)R^(D).
 4. The method of claim 1, wherein the compound is


5. The method of claim 1, wherein the particle or polymer furthercomprises a reporting agent.
 6. The method of claim 1, wherein thefluorescence image is obtained by fluorescence microscopy.
 7. The methodof claim 1, wherein the optical sensor conjugate is used in an in vitroassay.
 8. The method of claim 1, wherein the subject is a mammal.
 9. Themethod of claim 1, wherein the reactive oxygen, reactive nitrogen, orboth are associated with inflammation.
 10. The method of claim 9,wherein the inflammation is associated with tissue injury.
 11. Themethod of claim 9, wherein the reactive oxygen, reactive nitrogen, orboth are associated with Alzheimer's disease, atherosclerosis, cancer,stroke, inflammatory bowel disease, or organ transplantation.